Cyclic voltammetric study of the redox system of glutathione using the disulfide bond reductant tris(2-carboxyethyl)phosphine

The stabilization of the reduction state of proteins and peptides is very important for the monitoring of protein-protein, protein-DNA and protein-xenobiotic interactions. The reductive state of protein or peptide is characterized by the reactive sulfhydryl group. Glutathione in the reduced (GSH) and oxidized (GSSG) forms was studied by cyclic voltammetry. Tris(2-carboxyethyl)phosphine (TCEP) as the disulfide bond reductant and/or hydrogen peroxide as the sulfhydryl group oxidant were used. Cyclic voltammetry measurements, following the redox state of glutathione, were performed on a hanging mercury drop electrode (HMDE) in borate buffer (pH 9.2). It was shown that in aqueous solutions TCEP was able to reduce disulfide groups smoothly and quantitatively. The TCEP response at -0.25 V vs. Ag/AgCl/3 M KCl did not disturb the signals of the thiol/disulfide redox couple. The origin of cathodic and anodic signals of GSH (at -0.44 and -0.37 V) and GSSG (at -0.69 and -0.40 V) glutathione forms is discussed. It was shown that the application of TCEP to the conservation of sulfhydryl groups in peptides and proteins can be useful instrument for the study of peptides and proteins redox behavior.     

Selective detection of the reduced form of glutathione (GSH) over the oxidized (GSSG) form using a combination of glutathione reductase and a Tb(III)-cyclen maleimide based lanthanide luminescent 'switch on' assay

The synthesis of a novel Tb(III) luminescent probe for the detection of thiols is presented. The probe 1.Tb, possessing a maleimide moiety, as its sulfhydryl acceptor, was poorly emitting in aqueous pH 7 solution in the absence of a thiol. However, upon addition of thiols such as glutathione (GSH), large enhancements were observed, particularly within the physiological pH range. In contrast no enhancements were observed in the presence of the oxidized form of glutathione (GSSG), except in the presence of the enzyme glutathione reductase and NADPH which enabled 1.Tb to be used to observe the enzymatic reduction of GSSG to GSH in real time.     

Study of Oxidation of Glutathione by Capillary Electrophoresis

Glutathione (GSH) occurs widely in animal and human's tissues, and protects cells by changing into reversible oxidized glutathione (GSSG) when cells meet with oxidants, such as hydrogen peroxide (H2O2) and lipid peroxide. They are of great importance in a variety of diseases, which possess an oxidative etiology. The conversion of GSH to GSSG is widely recognized as a reliable index of oxidative stress1. There are some reports about determination of GSH and GSSG by high performance liq…     

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from 'total GSH'. The linear range and limit of detection for both analytes were 7.5 × 10(-7) to 1 × 10(-5) M, and 5 × 10(-7) M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL(-1)), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL(-1) glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL(-1) (p < 0.05), which was rationalised by considering measurements of H(2)O(2) and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL(-1) glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.     

Determination of cellular redox status by stable isotope dilution liquid chromatography/mass spectrometry analysis of glutathione and glutathione disulfide

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.     

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

A simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG.

Simultaneous analysis of oxidized and reduced glutathione in cell extracts by capillary zone electrophoresis

Glutathione (GSH) and glutathione disulfide (GSSG) levels in cells constitute a thiol redox system. They can be used as an indicator of oxidative stress of the cell. In this study, a capillary zone electrophoresis (CZE) method is described that enables quantitation of GSH and GSSG from cellular extracts. The CZE buffer used was 20 mM ammonium acetate containing 5% (v/v) acetic acid at pH 3.1 in conjunction with a polybrene coated capillary operated in reverse polarity mode. Effects of different acids used to prepare cell samples were investigated on CZE performance. The acids include meta phosphoric acid (MPA), trichloroacetic acid (TCA), phosphoric acid (PA) and sulfosalicylic acid (SSA) and are used to stabilize GSH and GSSG before performing CZE analysis. The method features a limit of detection of 4 microM and a limit of quantitation of 12 microM for both GSSG and GSH and recoveries of 94% for GSH and 100% for GSSG. Quantitative analysis of GSSG and GSH in HaCaT cell extracts (5% SSA, w/v) was performed with this method and changes in the ratio of GSH to GSSG in N-ethylmaleimide treated cell sample was observed by comparing with control cell samples.     

The Copper(II)‐Catalyzed Oxidation of Glutathione

The kinetics and mechanisms of the copper(II)‐catalyzed GSH (glutathione) oxidation are examined in the light of its biological importance and in the use of blood and/or saliva samples for GSH monitoring. The rates of the free thiol consumption were measured spectrophotometrically by reaction with DTNB (5,5′‐dithiobis‐(2‐nitrobenzoic acid)), showing that GSH is not auto‐oxidized by oxygen in the absence of a catalyst. In the presence of Cu²⁺, reactions with two timescales were observed. The first step (short timescale) involves the fast formation of a copper–glutathione complex by the cysteine thiol. The second step (longer timescale) is the overall oxidation of GSH to GSSG (glutathione disulfide) catalyzed by copper(II). When the initial concentrations of GSH are at least threefold in excess of Cu²⁺, the rate law is deduced to be ?d[thiol]/dt=k[copper–glutathione complex][O₂]^0.5[H₂O₂]^?0.5. The 0.5^th reaction order with respect to O₂ reveals a pre‐equilibrium prior to the rate‐determining step of the GSSG formation. In contrast to [Cu²⁺] and [O₂], the rate of the reactions decreases with increasing concentrations of GSH. This inverse relationship is proposed to be a result of the competing formation of an inactive form of the copper–glutathione complex (binding to glutamic and/or glycine moieties).     

Accurate quantification of the redox-sensitive GSH/GSSG ratios in the yeast Pichia pastoris by HILIC–MS/MS

A novel method for the simultaneous quantification of both glutathione (GSH) and its oxidized form glutathione disulfide (GSSG) by hydrophilic interaction chromatography–MS/MS has been developed and is critically discussed. Internal standardization based on isotopically labeled standards for both analytes is an absolute prerequisite for accurate quantification of this redox pair. Hence, a highly efficient and selective miniaturized procedure for the synthesis of isotopically labeled GSSG from commercially available glutathione-(glycine-¹³C₂,¹⁵N) was established using H₂O₂ as oxidant and NaI as catalyst. Moreover, a tool is presented to monitor and hence uncover artifactual GSSG formation due to oxidation of GSH during sample preparation, which is the main source of systematic error in GSSG analysis. For this purpose, we propose to monitor the oxidation product formed by reaction of naturally occurring GSH with the isotopically labeled GSH used as internal standard. For the determination of GSH/GSSG ratios in yeast, different extraction methods based on (1) hot extraction with aqueous, acidic, or organic solvents, (2) mechanical cell lysis, and (3) extraction at subambient temperature were investigated in terms of recovery, extraction efficiency, and artifactual formation of GSSG. Total combined uncertainties of as low as 25–30 % (coverage factor?=?2) for the determination of GSH/GSSG ratios without derivatization were made possible by the addition of the internal standards early in the analytical procedure (before extraction) and immediate analysis of the analytes.     

Disposable Amperometric Sensors for Thiols with Special Reference to Glutathione

The antioxidant ‘reduced glutathione’ tripeptide is conventionally called glutathione (GSH). The oxidized form is a sulfur‐sulfur linked compound, known as glutathione disulfide (GSSG). Glutathione is an essential cofactor for antioxidant enzymes; it provides protection also for the mitochondria against endogenous oxygen radicals. The ratio of these two forms can act as a marker for oxidative stress. The majority of the methods available for estimation of both the forms of glutathione are based on colorimetric and electrochemical assays. In this study, electrochemical sensors were developed for the estimation of both GSH and GSSG. Two different types of transducers were used: i) screen‐printed three‐electrode disposable sensor (SPE) containing carbon working electrode, carbon counter electrode and silver/silver chloride reference electrode; ii) three‐electrode disposable system (CDE) consisting of three copper electrodes. 5,5′‐dithiobis(2‐nitrobenzoic acid) (DTNB) was used as detector element for estimation of total reduced thiol content. The enzyme glutathione reductase along with a co‐enzyme reduced nicotinamide adenine dinucleotide phosphate was used to estimate GSSG. By combining the two methods GSH can also be estimated. The detector elements were immobilized on the working electrodes of the sensors by bulk polymerization of acrylamide. The responses were observed amperometrically. The detection limit for thiol (GSH) was less than 0.6 ppm when DTNB was used, whereas for GSSG it was less than 0.1 ppm.     

Thiol cofactors for selenoenzymes and their synthetic mimics

The importance of selenium as an essential trace element is now well recognized. In proteins, the redox-active selenium moiety is incorporated as selenocysteine (Sec), the 21st amino acid. In mammals, selenium exerts its redox activities through several selenocysteine-containing enzymes, which include glutathione peroxidase (GPx), iodothyronine deiodinase (ID), and thioredoxin reductase (TrxR). Although these enzymes have Sec in their active sites, they catalyze completely different reactions and their substrate specificity and cofactor or co-substrate systems are significantly different. The antioxidant enzyme GPx uses the tripeptide glutathione (GSH) for the catalytic reduction of hydrogen peroxide and organic peroxides, whereas the larger and more advanced mammalian TrxRs have cysteine moieties in different subunits and prefer to utilize these internal cysteines as thiol cofactors for their catalytic activity. On the other hand, the nature of in vivo cofactor for the deiodinating enzyme ID is not known, although the use of thiols as reducing agents has been well-documented. Recent studies suggest that molecular recognition and effective binding of the thiol cofactors at the active site of the selenoenzymes and their mimics play crucial roles in the catalytic activity. The aim of this perspective is to present an overview of the thiol cofactor systems used by different selenoenzymes and their mimics.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

Investigation of Cd(II), Pb(II) and Cu(I) complexation by glutathione and its component amino acids by ESI-MS and size exclusion chromatography coupled to ICP-MS and ESI-MS

The elucidation of structures of glutathione (GSH) complexes play an important role in the fundamental understanding of biochemical pathways of metal ion deactivation in plants. This article attempts to feature key studies for stoichiometry of metal complexes with glutathione and its constituent amino acids to obtain a better understanding of the different metal affinities of the complexation sites of glutathione. The SEC-ICP-MS experiments have indicated that oxidation process of glutathione was accelerated by metal ion presence in following order Cu⁺, Pb²⁺ and Cd²⁺. The redox activity of metal ions was confirmed by ESI-MS experiments, which allowed to observe formation of glutathione disulphide (GSSG) in time. The stoichiometry of Cd²⁺, Cu⁺ and Pb²⁺ complexes with GSH was defined by observing the isotope pattern of investigated metals and hydrogen loss or transfer during binding. The complexes with metal bound to sulphur of 1:1 and 1:2 stoichiometry were found in case of cadmium and lead. The number of hydrogen atoms lost during metal binding and the SEC-ESI-MS results allowed to elucidate that copper is bound by GSSG in ratio 1:1 and 1:2. Additionally, size exclusion chromatography coupled to electrospray MS allowed to differentiate more stable complexes from weak ones that could be created in the gas phase.     

Disulfide bond cleavage induced by a platinum(II) methionine complex

The cleavage of a disulfide bond and the redox equilibrium of thiol/disulfide are strongly related to the levels of glutathione (GSH)/oxidized glutathione (GSSG) or mixed disulfides in vivo. In this work, the cleavage of a disulfide bond in GSSG induced by a platinum(II) complex [Pt(Met)Cl2] (where Met = methionine) was studied and the cleavage fragments or their platinated adducts were identified by means of electrospray mass spectrometry, high-performance liquid chromatography, and ultraviolet techniques. The second-order rate constant for the reaction between [Pt(Met)Cl2] and GSSG was determined to be 0.4 M(-1) s(-1) at 310 K and pH 7.4, which is 100- and 12-fold faster than those of cisplatin and its monoaqua species, respectively. Different complexes were formed in the reaction of [Pt(Met)Cl2] with GSSG, mainly mono- and dinuclear platinum complexes with the cleavage fragments of GSSG. This study demonstrated that [Pt(Met)Cl2] can promote the cleavage of disulfide bonds. The mechanistic insight obtained from this study may provide a deeper understanding on the potential involvement of platinum complexes in the intracellular GSH/GSSG systems.     

Chip-based SALDI-MS for rapid determination of intracellular ratios of glutathione to glutathione disulfide

Alterations in the ratio of glutathione(GSH) to glutathione disulfide(GSSG) reveal the cell living state and are associated with a variety of diseases. In this study, an Au NPs grafted nanoporous silicon chip was used for surface assisted laser desorption ionization-mass spectrometry(SALDI-MS) detection of GSH. Due to the bond interaction between thiol of GSH and Au NPs modified on the chip surfaces, GSH could be captured from the complex cellular lysate. Meanwhile, the composite nanostructures of Au NPs grafted porous silicon surface presented good desorption/ionization efficiency for GSH detection. The GSH levels in different tumor cells were successfully detected. Chip-based SALDI-MS was optimized for quantification of intracellular GSH/GSSG ratio changing under drug stimulation in liver tumor cells, GSSG was reduced to GSH by reductant of tris(2-carboxyethyl)phosphine(TCEP) and isotope-labeling GSH was as an internal standard. It was found that the increasing concentration of drug irinotecan and hypoxia culture condition caused the rapid consumption of GSH and a decrease of GSH/GSSG ratio in liver tumor cells. The developed SALDI-MS method provided a convenient way to accurately measure and rapidly monitor cellular GSH value and the ratios of GSH/GSSG.     

Kinetics and Equilibria of the Thiol/Disulfide Exchange Reactions of Somatostatin with Glutathione

Rate and equilibrium constants are reported for the thiol/disulfide exchange reactions of the peptide hormone somatostatin with glutathione (GSH). GSH reacts with the disulfide bond of somatostatin to form somatostatin-glutathione mixed disulfides (Cys(3)-SH, Cys(14)-SSG and Cys(3)-SSG, Cys(14)-SH), each of which can react with another molecule of GSH to give the reduced dithiol form of somatostatin and GSSG. The mixed disulfides also can undergo intramolecular thiol/disulfide exchange reactions to re-form the disulfide bond of somatostatin or to interconvert to the other mixed disulfide. Analysis of the forward and reverse rate constants indicates that, at physiological concentrations of GSH, the intramolecular thiol/disulfide exchange reactions that re-form the disulfide bond of somatostatin are much faster than reaction of the mixed disulfides with another molecule of GSH, even though the intramolecular reaction involves closure of a 38-membered ring. Thus, even though the disulfide bond of somatostatin is readily cleaved by thiol/disulfide exchange, it is rapidly reformed by intramolecular thiol/disulfide exchange reactions of the somatostatin-glutathione mixed disulfides. By comparison with rate constants reported for analogous reactions of model peptides measured under random coil conditions, it is concluded that disulfide bond formation by intramolecular thiol/disulfide exchange in the somatostatin-glutathione mixed disulfides is not completely random, but rather it is directed to some extent by conformational properties of the mixed disulfides that place the thiol and mixed disulfide groups in close proximity. A reduction potential of -0.221 V was calculated for the disulfide bond of somatostatin from the thiol/disulfide exchange equilibrium constant.     

Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection

A method for the simultaneous quantification of reduced and oxidized glutathione in human plasma employing a two-dimensional chromatographic system with parallel porous graphitized carbon (PGC) columns coupled with fluorescence (FLD) and coulometric electrochemical detection (ED) has been developed. Post-sampling oxidation of reduced glutathione (GSH) was prevented by derivatizing the -SH group with monobromobimane (MBB) and the glutathione-bimane adduct (GSMB) was detected by FLD. Oxidized glutathione (GSSG) was detected by ED optimized to give lowest possible limits of detection (LOD). The method is fully validated and is currently used for determination of GSH, GSSG and its redox potential in different clinical studies.     

Voltammetric Investigation of Zinc Release from Metallothioneins Modulated by the Glutathione Redox Couple and Separated with a Porous Membrane

Glutathione (GSH), in addition to serving as a redox buffer in cellular environment, has been suggested as a modulator in metal regulation and homeostasis by metallothioneins (MTs). The interactions of MTs with both GSH and its oxidized form GSSG have been shown to govern the direction of metal transfer. Common methods for the determination of zinc release from MTs modulated by GSH/GSSG either involve radioactive species or enzymes or are labor‐intensive. In this study, upon separation of Zn²⁺ from the reaction mixture of MTs and GSH with a centrifugal filter membrane, differential pulse voltammetry (DPV) was used for the Zn²⁺ quantification. The same approach is extended to the studies of metal transfer between Zn₇MT with a GSH/GSSG mixture and that between Zn₇MT with GSSG. The concomitant conversion between the free thiol and disulfide bonds was confirmed with UV‐vis spectrophotometry. The results demonstrate that GSSG, GSH, and the GSH/GSSG mixture all modulate zinc release from Zn₇MT. The percentage of zinc release increases in the order of GSH, GSSG, and the GSH/GSSG mixture. The new approach is demonstrated to be well suited for investigation of redox regulation of MT and its reaction with zinc‐containing enzymes.     

Ratios of biliary glutathione disulfide (GSSG) to glutathione (GSH): a potential index to screen drug-induced hepatic oxidative stress in rats and mice

Hepatotoxicity of drug candidates is one of the major concerns in drug screening in early drug discovery. Detection of hepatic oxidative stress can be an early indicator of hepatotoxicity and benefits drug selection. The glutathione (GSH) and glutathione disulfide (GSSG) pair, as one of the major intracellular redox regulating couples, plays an important role in protecting cells from oxidative stress that is caused by imbalance between prooxidants and antioxidants. The quantitative determination of the GSSG/GSH ratios and the concentrations of GSH and GSSG have been used to indicate oxidative stress in cells and tissues. In this study, we tested the possibility of using the biliary GSSG/GSH ratios as a biomarker to reflect hepatic oxidative stress and drug toxicity. Four compounds that are known to alter GSH and GSSG levels were tested in this study. Diquat (diquat dibromide monohydrate) and acetaminophen were administered to rats. Paraquat and tert-butyl hydroperoxide were administered to mice to induce changes of biliary GSH and GSSG. The biliary GSH and GSSG were quantified using calibration curves prepared with artificial bile to account for any bile matrix effect in the LC–MS analysis and to avoid the interference of endogenous GSH and GSSG. With four examples (in rats and mice) of drug-induced changes in the kinetics of the biliary GSSG/GSH ratios, this study showed the potential for developing an exposure response index based on biliary GSSG/GSH ratios for predicting hepatic oxidative stress.     

Synthesis of Peptide Cysteine Dimedone Using Fmoc-Cys(Dmd)-OH: Glutathione Cysteine Dimedone as a Probe in Investigating the Sulfenic Acid Mediated Oxidation of Glutathione

Dimedone is the most widely used chemical probe for detection of cysteine sulfenic acid in peptides and proteins. The reaction of dimedone with cysteine sulfenic acid results in the formation of unique cysteine dimedone motif containing thioether bridge. Based on the structure of cysteine dimedone residue in polypeptide, a new building block of Fmoc-Cys(Dmd)-OH was developed for solid phase synthesis of peptide cysteine dimedone. Mass spectrometric sequencing of synthetic peptides have confirmed successful incorporation of cysteine dimedone in peptide chain using HBTU/HOBt as a coupling agent. The new method permits synthesis of peptides containing both cysteine thiol and cysteine dimedone in the same sequence which was difficult to achieve by conventional methods. The synthetic peptide of glutathione cysteine dimedone was used as a standard in probing the air-mediated oxidation of thiol to disulfide form of glutathione. The co-elution of standard peptide and reaction mixture of oxidation of glutathione in presence of dimedone using RP-HPLC have confirmed the formation of glutathione cysteine sulfenic as an intermediate in the air-mediated oxidation of glutathione. The synthetic peptides of cysteine dimedone may find application in the field of redox proteomics and generation of antibodies against modified cysteine residue.